1. Field of the Invention
The present invention relates to the functional expression of eukaryotic cytochrome P450s in bacterial cells, genetic constructs for effecting such expression and methods employing these bacteria and/or the recombinant enzyme so produced, e.g., as bioreactors for effecting the metabolism of cytochrome P450 substrates.
2. Description of the Related Art
The cytochrome P450 superfamily includes multiple molecular forms of enzymes which catalyze monooxygenase reactions of a wide variety of endogenous as well as exogenous substrates (Lu, et al., 1980). Each form of these hemoproteins exhibits a unique substrate specificity. The cytochrome P450 superfamily of enzymes participate, for example, in the metabolism of steroids (Hall, 1980), eicosanoid, fatty acids (Kupfer, 1980), and bile acids (Hansson and Wikvall, 1980), as well as exogenous substrates such as drugs, insecticides, and chemical carcinogens (Gelboin, 1980). Comparison of several forms of cytochrome P450 whose primary structures have so far been reported indicates that they are structurally related to one another and possibly derived from a common ancestor (Gotoh, et al, 1983).
Microsomal cytochrome P450s are integral membrane hemoproteins which derive reducing equivalents from NADPH by means of a membrane bound flavoprotein oxido-reductase (NADPH-cytochrome P450 reductase). These so-called mixed-function oxidases activate molecular oxygen so as to insert one atom into a lipophilic substrate and the other atom into water Cytochrome P450 and P450-like enzymes are ubiquitous in nature, being found in a broad range of eukaryotes as well as bacteria. In fact, many of the bacterial enzymes are similar to a certain degree to the P450 enzymes found in mammals. For example, in certain soil bacteria (i.e., Pseudomonas putida), the oxygenation of camphor involves an enzyme termed cytochrome P450cAM which acts in concert With an FAD-containing flavoprotein, putida redoxin reductase and an iron sulfur protein putida redoxin (Katagira, et al., 1968) Interestingly, the bacterial electron transfer system in P. putida is very similar to the one functional in the mitochondria of higher organisms. However, the electron transfer system of P. putida cannot support the functional transfer of electrons to either mitochondrial or microsomal P450s. Because of sequence similarities between the medically important mammalian microsomal P450 (Family IV) and the bacterial fatty acid monooxygenase from Bacillus megaterium (cytochrome P450.sub. BM3), a large amount of effort has been expended in characterizing this and other bacterial enzymes.
While a fair amount of research has been conducted in characterizing bacterial cytochrome P450s, their use in commercial applications, such as in bioreactors, chemical degradation and drug synthesis, has been quite limited. The primary reason for this is that the number of specific forms of bacterial P450s that have been well characterized is very small and the reactions that these enzymes catalyze are of limited biomedical or commercial value. In contrast, a vast number of eukaryotic and mammalian P450s have been characterized. More importantly, these enzymes catalyze reactions involved in drug, steroid and xenobiotic metabolism all of which have a direct impact on human health. This is unfortunate in that bacterial expression systems offer many advantages over expression in yeast and mammalian cells in that bacterial systems may be used to produce proteins in large quantities, at low cost, with unparalleled ease.
In contrast to the disadvantages associated with the use of bacterial cytochrome P450s, the eukaryotic enzymes offer many advantages, including metabolism of drugs and other molecules of importance to human health. Unfortunately, present eukaryotic systems for the production of eukaryotic cytochrome P450s are hampered by several serious drawbacks including low rates of production, the use of expensive tissue culture materials and facilities as well as the requirement for sophisticated methodologies, and extensive destruction of the enzyme by the expression host or even lack of biological activity.
Accordingly, there is a great need for the development of technology which would combine the advantages of bacterial expression with those of eukaryotic and mammalian cytochrome P450s. Although bacteria have demonstrated great utility in the expression of many prokaryotic and eukaryotic proteins, bacterial expression systems for cytochrome P450 have heretofore been limited to the soluble bacterial forms of this gene superfamily (Unger, et al., 1986). Although yeast (Oeda, et al., 1985), COS 1 (Zuber, et al., 1986) and virally infected eukaryotic cells (Battula, et al., 1987; Asseffa, et al., 1989) have been used as hosts for the heterologous expression of P450 molecules, each has limitations to their usefulness as expression systems. Unfortunately, the present understanding in the art is that eukaryotic cytochrome P450s simply cannot be expressed functionally in bacteria (Collin, et al., 1988).
Due to the existence of numerous disadvantages with current systems for the expression of biologically active cytochrome P450s, there exists a continuing need for the development of novel systems, particularly bacterial expression systems, which can be used to produce biologically active cytochrome P450 enzymes. There is a particular need for bacterial expression systems for expressing biologically active eukaryotic cytochrome P450s which incorporate the advantages of bacterial expression. The development of novel technology which addresses one or more of these disadvantages would have broad research and commercial applications including steroid and prostaglandin biosynthesis, as bioreactors, in drug development and characterization, and even environmental clean up.